Engineering a robust Pseudomonas putida KT2440 via promoter enhanced strategy for effective quorum quenching and biofilm suppression in Wastewater treatment systems

Quorum quenching (QQ) of signaling molecules plays a critical role in reducing biofilm and controlling membrane biofouling for sustained performance. The aiiO gene serves as a key microbial regulatory gene responsible for implementing quorum quenching. Appropriate expression of the aiiO gene can address key issues in wastewater treatment and avoid the excessive microbial load that results from overproduction. In this study, promoter-engineering strategy is used to enhance QQ effect in Pseudomonas putida KT2440 by modulating expression of the aiiO lactonase. Promoters of graded strength (p51, p46, pJ23119, and plac) were cloned upstream of aiiO and aiiO–gfp in the broad-host-range plasmid pBBR1-MCS2, enabling tunable, dose-dependent attenuation of acyl-homoserine lactone (AHL) signaling. In activated sludge systems, the p51-driven strain delivered the most pronounced effect, reducing biofilm formation by ∼50–60% relative to natural quorum quenching Rhodococcus BH4 after 84h, while concurrently lowering EPS secretion (51.42%) and weakening biofilm adhesion (28.5%). Importantly, p51-mediated aiiO expression imposed no detectable metabolic burden on the host KT2440, and QQ performance remained stable over extended cultivation, indicating genetic and phenotypic robustness suitable for long-term operation. Through targeted regulation of aiiO expression, this study achieved tunable quorum quenching activity in activated sludge systems, leading to reduced AHL-mediated biofilm formation, lower EPS secretion, and weaker biofilm adhesion, thereby providing a promising strategy for mitigating biofouling in wastewater treatment applications.